Tumor markers are continually being sought to aid in both the monitoring of and the in-vitro diagnosis of cancer, as is well known in the art. For example, commercial assays, such as the carcinoembryonic antigen and alpha-fetoprotein immunoassays, are available to detect the presence of certain tumor markers in biological fluids.
Serum secretory IgA(SIgA) has been reported in the literature as a tumor marker for various carcinomas. Some researchers report that assays for total SIgA are too non-specific for cancer and of no value for cancer detection, LoGerfo, P. et al., (1976) J. Surg. Res., 20:481. Other researchers showed that the measurement of total SIgA could be of value diagnostically in certain instances of carcinomas and chronic liver disease, Homburger, H. A., et al., (1984) A.J.C.P. 81:569; Kvale, D. et al., (1987) Cancer 59:203; Watanabe, T. et al., (1983) Otolaryngol Head Neck Surg 91:136. As a result, immunoglobulins or other glycoproteins that may be associated with cancer and other disease states, especially chronic hepatic disease, have been investigated for use in diagnostic assays.
No single tumor marker currently available for testing can detect all carcinomas or pre-cancerous stages of a potential carcinoma. Therefore, a number of assays may be used to diagnose and monitor these disease states in order to improve the accuracy of the diagnosis. One problem is that some tumor markers may not be detected because current assays are not sensitive or specific enough to do so and important diagnostic information is never obtained.
Another problem associated with producing a specific and sensitive immunoassay is that repeated attempts to develop antibodies to a very specific antigen can be unsuccessful, thereby making an immunoassay to a specific antigen impossible. Also, the production of antibodies, when possible, is an elaborate and expensive process. The ability of a naturally occurring substance to replace antibodies in an assay would be beneficial.
In order to avoid using antibodies either partially or totally in an assay, and because they posses certain binding affinities, lectins have been employed in various assays. Lectins can recognize and bind to particular carbohydrate structures on the oligosaccharide moieties of glycoproteins. For instance, U.S. Pat. Nos. 4,389,392 and 4,571,382 to Adachi describe methods of using lectins which bind to glycoproteins with a terminal galactose (Beta 1-3 or Beta 1-4)-N-acetylglucosamine or terminal galactose (Beta 1-3 or Beta 1-4)-N-acetylgalactosamine in order to detect tumor associated glycolinkage containing substances. However, the methods described in these patents fail to use any immunological means to first specify which molecules are to be assayed, thereby losing a large measure of specificity.
PCT Application No. WO 87/00289 discloses a test method in which lectins are used to form complexes with soluble desialylated glycoproteins which are then contacted with a detecting antibody. A variant of the test method is disclosed where the desialylated glycoprotein forms a complex with a specific antibody and the labeled lectin is then contacted with the complex. In both cases, the antibody must be specific to the desialylated glycoprotein in order to selectively bind to the glycoprotein. Although the lectin is specific to a particular binding site on the oligosaccharide moiety of the desialylated glycoprotein, a substantial portion of the specificity of the test lies with the ability of the antibody to bind to the correct antigen.
In EPO Patent Application 0 043 359 a test method is described in which a solid phase coated with an antibody specific to the glycoprotein to be analyzed is contacted with a sample to form a complex, to which the labeled lectin is added. Again, the antibody must be specific to the glycoprotein. A similar assay configuration is described by Pekelharing, J. M., et al., (1987) Anal. Bio. 165:320.
The oligosaccharide moieties of glycoproteins are attached to the protein moiety by O-glycosidic or N-glycosidic bonds. Carbohydrate structures of glycoprotein oligosaccharides synthesized by tumor cells are often different from the carbohydrate structures in the normal cell counterpart, thereby making such glycoproteins potential tumor markers. Many of these tumor markers have been identified as mucin glycoproteins. Mucins are high molecular weight glycoproteins characterized by unusually high carbohydrate content and carbohydrate side chains that are linked by O-glycosidic bonds to the protein chains. IgA.sub.1, but not IgA.sub.2, also contains carbohydrate side chains that are linked to the protein chain by O-glycosidic bonds. The O-linkage occurs between the hydroxyl groups of serine or threonine of the polypeptide and the reducing ends of N-acetylgalactosamine of the oligosaccharide. Certain lectins recognize and bind to the D galactose Beta (1-3)-N-acetylgalactosamine (DGalBeta(1 3)DGalNAc) oligosaccharide structure O-linked to the polypeptide chains of IgA.sub.1 and mucin.
Mucins and IgA.sub.1 are the major glycoproteins in serum and body fluids with this O-linkage; the vast majority of serum glycoproteins have N-glycosidic linkages. These N-linkages occur between the amide nitrogen of asparagine and the reducing end of N-acetylglucosamine.
Oligosaccharide moieties of O-linked glycoproteins may be altered in cancer patients due to tumor cell necrosis and the release of glycosyltransferases and glycosidases in the vicinity of tumors. The glycoproteins IgA.sub.1 and mucin contain O-linked oligosaccharides and can serve as substrates for the released glycosyltransferases and glycosidases. When glycosidases and glycosyltransferases are released from tumors, the O-linked carbohydrate structure of the IgA.sub.1 molecule may be altered.
Unaltered IgA.sub.1 does contain DGalBeta(1-3)DGalNAc structures accessible to lectin binding, resulting in low levels or background normal levels of these O-linked carbohydrate side chains. However, when the IgA.sub.1 molecule is altered by glycosidases the number of these DGalBeta(1-3)DGalNAc structures accessible to lectin binding increases.
It would be advantageous to be able to detect these cancer-altered IgA.sub.1 immunoglobulins or to detect a change in the proportion of IgA.sub.1 to IgA.sub.2 by measuring the O-linked carbohydrate side chains on the IgA.sub.1 molecules with a high level of specificity. Particularly advantageous would be an immunoassay method that uses lectins and not an antibody to detect these side chains.